Cyclin-dependent kinases (Cdks) drive the events of the eukaryotic cell cycle. Cdks are thought to act by phosphorylating a large number of protein substrates in the cell, but few of these substrates have been identified. In the proposed studies, a novel biochemical strategy will be used to perform large scale searches for Cdk substrates in budding yeast and vertebrates. Our preliminary studies have led to the identification of about 200 Cdkl substrates in budding yeast, and in the first aim of the proposed work a subset of these substrates will be analyzed in detail. Other preliminary work has revealed that the identity of the associated cyclin subunit has a profound effect on the rate at which Cdkl phosphorylates many yeast substrates, and the second aim of the proposed studies will be to explore the molecular basis of this cyclin specificity and its physiological function in the cell. Methods will also be developed for the systematic identification and characterization of large numbers of Cdk substrates in vertebrate cells. Finally, a 'chemical genetic' approach will be employed to construct mice and mouse cell lines in which Cdkl or Cdk2 can be inhibited with high specificity, allowing rigorous analysis of their function in normal cell biology and in tumors. These studies promise to provide a wide range of new insights into the basic mechanisms by which Cdks drive cell-cycle events. Such insights are an important step toward a better understanding of the chromosome segregation defects and other cell-cycle defects that characterize the cancer cell.